There is a general perception that pathogenic bacteria are increasingly infecting the food supply, air supply and recreation and drinking water supply. In the last few decades, the incidence of microbial infection and water-borne disease has significantly increased worldwide. The contamination by undesirable bacteria in foodstuffs, air, and water represents a significant threat to public health. Monitoring efforts rely on conventional microbiological techniques to detect the presence of bacteria, typically including the growth of bacteria on nutrient media. The size, color and morphology of bacterial colonies are among the criteria used to identify the species present. In many cases, several different media must be employed in order to discriminate one species from another. The time necessary to carry out the many steps to achieve this result may take up to several days and require a highly trained technician. Conventional bacterial identification and confirmation techniques utilizing membrane filtration require culturing of a specimen on selective media with selection of potential colony types based on morphology and specific color, etc., to make a presumption, followed by a growth on a non-elective enrichment medium which is then transferred to a carbohydrate and pH indicator panel for confirmation. For certain membrane filtration procedures, the complete process can take several days.
In view of the increasing demand for clean water, food and air, it is essential to rapidly determine pathogen viability and growth potential prior to and after bacterialcidal treatment. Improvements are necessary in the specificity and simplicity of microbiological detection and management by developing quantitative indicators of parthenogenic bacterial viability. In recognition of the present time limitations for microbial detection, there is a need to simplify currently used methods and develop enhanced procedures for detection of viable pathogens. Current methods for determining water quality have been limited by multiple culture methods and the time required for the sampled bacteria to multiply. Efforts aimed at shortening that period have succeeded at reducing the time required from two days to as little as several hours, depending on the type of data required.
There are currently three accepted assay formats for the detection of parthenogenic bacteria in environmental samples: Multiple Tube Fermentation (MTF); Membrane Filtration (M) and Presence-Absence (PA). Based in part on technology developed in the 1920's, total and fecal coliform tests can require 24 to 72 hours to complete and rely on nonspecific bacterial biomass growth as the key indicator of fecal contamination.
Fecal coliforms are those coliform bacteria that are presumed from the feces of warm-blooded animals. Human fecal coliform bacteria which are primarily Escherichia coli (E. coli) also ferment lactose but at a higher temperature (44.5.degree. C.). E. coli are commonly found in the intestinal track of humans and animals but are not usually long term inhabitants of aquatic systems. Differentiation depends on an enrichment of the medium and an elevated incubation temperature of 44.5.degree..+-.0.2.degree. C. The presence of this species is an accepted indicator of the microbiological quality of water for drinking, for recreation, as well as of food.
Several possible rapid detection procedures for E. coli have been described in the art. For example, isotopic fecal detection tests have been shown to required as little as one hour, see Dange, V., Jothikumar, N., Khanna, P., "One hour portable test for drinking waters" Water Res 22:133137, 1988; Reasoner D.J., Geldreich, E. E., "Rapid Detection of Water-borne Fecal Coliforms by .sup.14 CO.sub.2 Release", Mechanizing Microbiology, Sharpe and Clark ed., 1978; Reasoner, D.J., Blannon, J.C., Geldreich, E.E., "Rapid Seven-hour Fecal Coliform Test", Appl Environ Microbiol., 38:229-236, 1979. Although these procedures are specific and fast, their disadvantages include the sophistication of the instruments required as well as the use of radio-active materials.
The detection of E. coli from environmental samples using the DNA hybridization and more recently Polymerase Chain Reaction shows technical promise (Bej et al., "Detection of Escherichia coli and Shigella sp. in water by using the Polymerase Chain Reaction and Gene Probes for Uid", Appl. Environ. Microbiol., 57(4): 1013-7, 1991). However, at present, the level of technical skill, specialized equipment and time required has dictated further development of appropriate and simpler methods.
Hydrolyzable substrates are dye moieties that are blocked in their initial condition and when cleaved during an enzyme hydrolysis step, provide chromogenic or fluorogenic signals. The determination of hydrolyric enzymes has been shown to be useful in the detection of certain species of bacteria. These methods to identify specific bacteria include the use of specific chromogenic or fluorogenic enzyme substrates and dyes, see Babb et al, U.S. Pat. No. 4,812,409; Hansen, W., Yourassowsky, E., "Detection of beta-glucuronidase in Lactose-fermenting Members of the Family Enterobacteriaceae and its Presence in Bacterial Urine Cultures", J. Clin. Micro., 20(4):1177-1179, 1984. A color change when cleaving a specific dye moiety would indicate the presence of a particular enzyme. Ideally, if there were one unique enzyme for each bacterial species of interest, one could determine its presence by monitoring the color change of the growth medium containing the substrate specific for that enzyme. This situation exists in no bacterial group described thus far, and is the focus of intense interest. It is commonly known that several bacterial species may share sets of enzymes. For example, E. coli produce several enzymes which may metabolize a number of substrates linked to a chromogen. However, other bacterial species (i.e., Cornybacterium, Shigella, etc.) also produce these and other enzymes. While imperfect, the use of single enzyme discrimination is permitted as part of the national primary water regulations to distinguish total and fecal coliforms (Rice, E.W., Allen, M.J., Brenner, D.J., Edberg, S.C., "Assay for beta-glucuronidase in Species of the Genus Escherichia and its Applications for Drinking-water Analysis", Appl. Environ. Microbiol., 57(2): 592-3, 1991). In addition, many substrates previously proposed become yellow, which is a color difficult to distinguish from normal bacteria biomass.